E. coli and Their Human Environment

Objectives:

Students will be able to observe how environmental changes (antibiotics) affect the growth of E.coli.

Students will be able to use the "F" test for statistical analysis.

Background:

E. coli is a comman rod-shaped intestinal bacterium. It lives in a mutualistic relationship with humans where it benefits from the warmth, shelter, and nutrients provided by the colon and in return makes vitamin K and sodium. It also serves as a competitive inhibitor of pathogenic bacteria.
E. coli does not cause disease or discomfort but does produce 400ml to 500ml of gas per day. Prolonged treatment with antibiotics may reduce the population of E. coli within the intestines.

Materials:

Petri dish (containing nutrient agar)

Wax pencil

E. coli culture-broth

Cotton swab

Scotch tape

Forceps

Ruler

antibiotics (four different kinds)

Incubator (37 degrees)

Software for computing "F" test

Data table of distribution of "F" values (F-values looked up by teacher or arrived at through use of appropriate statistics package).

Computer

Procedure:

DO NOT OPEN THE PETRI DISH!! Turn the dish upside down so the agar is on the top.

Using a wax pencil divide the bottom of the Petri dish into 4 equal sections. Number them 1 through 4 and place your initials towards the outer edge.

Turn the dish right side up. Take a cotton swab and dip it into the bacteria culture. Use caution for we do not want to contaminate the room or ourselves with bacteria.

Remove the top of the Petri dish and "sweep" the entire surface of the agar with the wet cotton swab. Turn the dish 90 degrees. Dip the cotton swab into the bacteria again and "sweep" the entire surface of the agar with more bacteria.

Place the cotton swab in the appropriate disposal container. Replace the top of the Petri dish.

Using forceps, remove one of the paper circles from the antibiotic container labeled #1. Place the circle in the center of section one of the Petri dish. Press it gently onto the agar. Place antibiotic #2 on section 2, antibiotic #3 on section 3, and antibiotic #4 0n section 4. Record the names of your antibiotics on your data table.

Your teacher will place a clean uncontaminated circle in another Petri dish that has been swept with bacteria.

Tape your Petri dish shut and place it in the incubator upside down (agar side up).

Disinfect your desk and wash your hands when done.

Next lab period:

Obtain your Petri dish from the incubator. Leave it upside down and do not open.

Measure the diameter of any clear areas (inhibition zones) around the paper circles (in mm) and record in the data table under your group letter.

If no clear area forms around the paper than measure the diameter of the paper circle (mm) and record in your data table.

Drawing:

Attach a drawing for the results of your experiment and label it. When you are done with your Petri dish give it to your instructor for proper disposal. Repeat procedure 9.

Statistical Analysis:

Complete the following data table:

Section

Anti-biotic

Diameter(mm) Each Group

Ave.

a

b

c

d

e

f

g

h

1

2

3

4

It is difficult, if not impossible, to draw conclusions about an experiment without subjecting the data to statistical analysis. When two independent samples are being studied a "T" test is performed to compare the differences between the means of the two samples. When more than two independent samples are being studied, as with this lab, an "F" test is performed to compare the differences between the means of the samples.

The null hypothesis for this experiment would be that the results you obtained were due to chance alone and not to the antibiotics. To test the null hypothesis the "F" test is used. There is software available (ex. Biostat) that will compute your "F" test. Your "F" value should than be compared to the table of distribution of "F" values. The software you used should give the degrees of freedom to help you find the table "F" value. If your "F" value is greater than the "F" value on the chart than you must reject the null hypothesis. This means that the antibiotics caused a statistical difference in the means of the different populations. If your "F" value was less than or equal to the "F" value on the chart than you must not reject the null hypothesis. This means your results were due to chance alone and not due to the antibiotics. When you subject your data to statistical analysis it allows you to make valid inferences about the results of your experiment.

Insert the appropriate software for computing the "F" test into the computer and run the "F" test.
Your "F" value is ________________.

Your "F" value should be compared to the table of "F" values (see Materials). The table "F" value is ________________ .

Questions:

What do the clear areas around the paper circles indicate?

What did the Petri dish your teacher prepared represent and what significance does it have?

Which antibiotic would you recommend for fighting this bacteria?

Which antibiotic would you least likely use to fight this bacteria?

What evidence do you have that the inhihition of microorganisms was due to the antibiotic on the circle and not the paper circle itself?

Bacteria belong to the KINGDOM

Observe the results of the class. Would it be a good idea to use the chemical that worked the best in class as an agent to fight other kinds of bacteria? Explain!

What is your calculated "F" value?

What is the acceptable "F" value on on the chart?

According to the results of the "F" test, is your data consistant with the null hypothesis that all sample means are equal or do you reject this hypothesis ? Explain!

Safety:

Students should wear safety glasses when working with E. coli.

Students should not open taped petri dishes.

Preparation:

The teacher should prepare agar plates (one per studentplus copntrol) one day in advance. The antibiotics canbe obtained free from a local hospital laboratory orcan be bought from a Biological Supply House.

Resource:

The Biostat program for statistical analysis was written by Steve Thompson. The address is: